Development of Recombinant Antibodies and Its Application in Immunomagnetic Separation-Based Rapid Detection of Vibrio cholerae in Aquatic Environments
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Cholera caused by Vibrio cholerae remains a major public health concern in many countries. The greatest obstacle to detection of V. cholerae contamination in drinking water or aquatic environments mainly relates to sample preparation steps, especially the enrichment step. In this study, immunomagnetic separation methods were developed based on sequence-defined recombinant antibodies (rAbs) against V. cholerae, then used for the specific and efficient enrichment of V. cholerae in water samples. Using the variable region genes of the anti-V. cholerae monoclonal antibodies (mAbs) 5F2, the full-length IgG rAbs (R5F2) were produced using mammalian human embryonic kidney 293T cells. Two antibodies, 5F2 and R5F2, were used to prepare immunomagnetic beads (IMBs), and their capture efficiencies (CEs) were evaluated. The results showed that 0.4 mg of 5F2-IMBs and R5F2-IMBs exhibited good CEs (96.0% and 75.9%, respectively) against V. cholerae within 40 min. The IMBs could still effectively capture V. cholerae in large-volume reaction systems (5 ml to 25 ml). The CEs of 5F2-IMBs and R5F2-IMBs ranged from 90.2% to 70.7% and 65.1% to 44.2%, respectively. Furthermore, 5F2-IMBs and R5F2-IMBs did not show significant cross-reactivity with other bacteria and exhibited high specificity. When R5F2-IMS was used in combination with quantitative real-time PCR, the detection limit was approximately 5 colony-forming units/25 ml after enrichment for 4 h. Our results suggest that the rAbs produced herein could provide useful alternatives to traditional hybridoma-based antibodies for accurate detection of V. cholerae in food safety and environmental monitoring.
Abstract
Cholera caused by Vibrio cholerae remains a major public health concern in many countries. The greatest obstacle to detection of V. cholerae contamination in drinking water or aquatic environments mainly relates to sample preparation steps, especially the enrichment step. In this study, immunomagnetic separation methods were developed based on sequence-defined recombinant antibodies (rAbs) against V. cholerae, then used for the specific and efficient enrichment of V. cholerae in water samples. Using the variable region genes of the anti-V. cholerae monoclonal antibodies (mAbs) 5F2, the full-length IgG rAbs (R5F2) were produced using mammalian human embryonic kidney 293T cells. Two antibodies, 5F2 and R5F2, were used to prepare immunomagnetic beads (IMBs), and their capture efficiencies (CEs) were evaluated. The results showed that 0.4 mg of 5F2-IMBs and R5F2-IMBs exhibited good CEs (96.0% and 75.9%, respectively) against V. cholerae within 40 min. The IMBs could still effectively capture V. cholerae in large-volume reaction systems (5 ml to 25 ml). The CEs of 5F2-IMBs and R5F2-IMBs ranged from 90.2% to 70.7% and 65.1% to 44.2%, respectively. Furthermore, 5F2-IMBs and R5F2-IMBs did not show significant cross-reactivity with other bacteria and exhibited high specificity. When R5F2-IMS was used in combination with quantitative real-time PCR, the detection limit was approximately 5 colony-forming units/25 ml after enrichment for 4 h. Our results suggest that the rAbs produced herein could provide useful alternatives to traditional hybridoma-based antibodies for accurate detection of V. cholerae in food safety and environmental monitoring.
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